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June 2017. A recent study from Dr. Rubio laboratory “Clarkson C., Antunes F.M. and Rubio M.E. (2016) Conductive hearing loss has long-lasting structural and molecular effects on pre- and post-synaptic structures of the auditory nerve in the cochlear nucleus J Neurosci 36(39):10214”, has been featured in Biomedical Advances (http://biomedical-advances.org) in the latest section of Neuroscience. Link: http://biomedical-advances.org/neuro-20175-18/

April 2017. A recent study from Dr. María Rubio in Brain Structure and Function investigated whether the number, density and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depended on the target cells. Using freeze-fracture replica immunogold labeling (FRIL) data showed that auditory nerve synapses on bushy and fusiform cells had similar numbers of AMPARs. However, there was a higher number and density of GluA3 subunits at auditory nerve synapses on bushy cells, whereas a higher number and density of GluA4 subunits were observed at auditory nerve synapses on fusiform cells. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, were concentrated at the center of the auditory nerve synapse on bushy cells. The central distribution of AMPARs was absent in GluA3-knockout mice, and gold particles were evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. In conclusion, the study showed that GluA3 and GluA4 subunits were distributed at auditory nerve synapses in a target-cell-dependent manner.

February 2017. A recent study by Dr. Sofía García-Hernández who is a postdoctoral trainee in Dr. María Rubio’s laboratory was published in Hearing Research. In this study, they investigated the role of the GluA3 AMPA receptor subunits in auditory processing by using wild type and GluA3 knockout mice. Auditory brainstem responses (ABR) were used to assess auditory function and electron microscopy to evaluate the ultrastructure of the endbulb of Held synapse. Since labeling for GluA3 subunit increases on endbulb synapses within the cochlear nucleus in response to transient sound reduction, they also investigated the role of GluA3 in experience-dependent changes in auditory processing. They found that the deletion of GluA3 led to impaired auditory signaling, early onset hearing loss and reduced numbers and sizes of postsynaptic densities of endbulb synapses. Additionally, the lack of GluA3 hampered ABR threshold recovery after transient sound reduction. The main conclusion of the study is that GluA3 is required for normal auditory signaling, normal ultrastructure of the endbulb of Held synapse in the cochlear nucleus and normal experience-dependent changes in auditory processing after transient sound reduction.

February 2017. Congratulations to Dr. Cat Weisz (former postdoc in the Kandler-lab and now Acting Chief, Section on Neuronal Circuitry, NIDCD) for receiving the 2017 Geraldine Dietz Fox Young Investigator Award. 

September 2016. A recent publication in the Journal of Neuroscience by Dr. Cheryl Clarkson and Flora Antunes, both postdoctoral trainees in Dr. Maria Rubio’s laboratory, investigated the response of the auditory brainstem during and after conductive hearing loss, the second most prevalent form of hearing loss. The study showed that transient monaural conductive hearing loss led to an increased in hearing thresholds, an increased central gain upstream the cochlear nucleus at level of the lateral lemniscus and to long-lasting pre- and post-synaptic structural and molecular effects at the endbulb of the Held synapse. Knowledge of the structural-molecular changes associated with decreased sensory experience, along with their potential reversibility, could be important for the treatment of hearing deficits, such as hyperacusis and chronic otitis media with effusion, which is prevalent in young children with language acquisition or educational disabilities.

August 2016. In a recent article in the Journal of Neuroscience the Tzounopoulos-lab descibed cell-specific effects ofAcetylcholine (ACh) in the auditory cortex. ACh is crucial for cognitive functions. Whereas in most cases the cellular circuits or the specific neurons via which ACh exerts its cognitive effects remain unknown, it is known that auditory cortex (AC) corticocollicular neurons projecting from layer 5B to the inferior colliculus are required for cholinergic-mediated relearning of sound localization after occlusion of one ear. Therefore, elucidation of the effects of ACh on the excitability of corticocollicular neurons will bridge the cell-specific and cognitive properties of ACh. These results suggest that cell-specific ACh-mediated persistent firing in corticocollicular neurons may represent a critical mechanism required for learning-induced plasticity in AC. Moreover, these results provide synaptic mechanisms via which ACh may mediate its effects on AC receptive fields.

May 2016. The National Institute on Deafness and Other Communication Disorders of the NIH awarded an Institutional Training Grant of one million dollars over five years to Dr. Karl Kandler and Dr. Bill Yates, who serve as the co-directors on this grant. This grant will support the research training of four pre- and postdoctoral students in auditory and vestibular neuroscience. 

May,  2016.  A recent article in the Journal of Neurophysiology by the Laboratory of Dr. Kandler, investigated the long-standing hypothesis that the polarity switch of inhibitory connections in the developing brain plays an important role in the remodeling of neuronal circuits, which is a prerequisite for normal brain function. The investigators addressed this question by comparing the developmental organization of excitatory and inhibitory connection in the auditory brainstem of normal mice and generically engineered mice, which lack this polarity switch. They found that these brains circuits develop normally even in the presence of a wrong polarity.  These unexpected results demonstrate that inhibitory connections in the auditory system can develop normally even if they elicit the wrong responses in their targets. The lead authors of this study are Dr. Hanmi Lee, a former graduate student and currently a Research Associate with Dr. Shatz in the Dept. of Neurobiology, Stanford University, and Dr. Eva Bach, a postdoctoral fellow supported by the DSF Charitable Foundation.  This project was in collaboration with Dr. Eric Delpire,  Vanderbilt University School of Medicine. Funding was provided by NIH grants from the National Institute on Deafness and Other Communication Disorders and the National Institute of Neurological Disorders and Stroke.

April 2016. In a recent article in the journal of Molecular Pharmacology, Dr. Manoj Kumar -- postdoctoral fellow with Dr. Tzounopoulos -- described the synthesis and evaluation of potent KCNQ2/3-specific potassium channel activators. This manuscript, which is the product of a close collaboration between the Wipf and the Tzounopoulos laboratories, illustrates the superiority of a novel compound, in terms of potency and specificity, over other potassium channel activators. Especially its superiority over retigabine, an FDA approved epilepsy drug, is a key finding that will be of interest to many. The manuscript highlights that the novel compound is a promising clinical candidate for treating or preventing neurological disorders associated with neuronal hyperexcitability, such as epilepsy and tinnitus. Due to the public relevance of the article, the Media Relations of our School of Medicine and UP Medical Center released a news article highlighting the significance of this work.

March 2016. Wai Lok (Cyrus) Tsang -- PSTP medical student with Dr. Thanos Tzounopoulos -- was awarded the prestigious Howard Hughes Medical Institute (HHMI) Medical Research Fellows award. This award will support his investigations towards understanding the molecular mechanisms of tinnitus. The benefits of participating in this fellowship program extend beyond the monetary reward and do not end after the fellowship years. During this award, Cyrus will have the opportunity to participate in the Medical Fellows regional group events, attend HHMI science meetings with HHMI investigators and interact with renowned physician-scientists.

January 2016. A recent article in the Journal of Neuroscience the Kandler-lab characterizes a new mechanism of neuronal communication between auditory synapses in the mammalian sound localization pathway. Using a combination of electrophysiological, anatomic and imaging approaches, Dr. Weisz, a former postdoc in the Kandler-lab and first author of the study, shows that the inhibitory neurotransmitter GABA can spill over between synaptic endings and stimulate neurotransmitter release. This excitatory action of GABA between inhibitory axon terminals may have important implications for the development and plasticity of auditory sound localization circuits and may play a role in the ability to precisely localize sound sources.

Congratulations to Dr. Cat Weisz on becoming the Acting Chief, Section on Neuronal Circuitry, Intramural Research Program of the NIDCD. In her new laboratory, Dr. Weisz, a former postdoctoral fellow in the Kandler-lab will investigate the neuronal circuits by which the brain influences the ear. Although these effect circuits (olivocochlear circuits) play an important role in hearing and in the protection of the ear from loud noise, their functioning is still poorly understood. For more information about the research in Dr. Weisz’s new lab click this link.

January 2016. In a recent article in the journal of Proceedings of the National Academy of Sciences (PNAS), Bopanna Kalappa, a postdoctoral fellow with Dr. Tzounopoulos, reported that synaptic zinc inhibits glutamate AMPA-type receptors (AMPARs). This effect is experience-dependent, because loud sound reduced presynaptic zinc levels in auditory brainstem synapses and abolished zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Ionotropic glutamate AMPARs play a fundamental role in normal function and plasticity of the brain, and they are also involved in many brain disorders. Despite the central role of AMPARs in neurobiology, the modulation of synaptic AMPA responses by endogenous modulators remains not well understood. Here, in three synapses found in two different brain areas, Kalappa et al., provide the first evidence, to our knowledge, that endogenous zinc is coreleased with glutamate and modulates the strength of synaptic AMPAR responses. Because in many neocortical areas more than 50% of excitatory presynaptic terminals contain zinc within their glutamatergic vesicles, these findings establish zinc as a general neuromodulator that allows for fine-tuning and plasticity of glutamatergic fast synaptic transmission in the brain.

September 2015. In a recent article in the journal Elife, Shuang Li, a graduate student in Dr. Tzounopoulos' laboratory reported the discovery of the mechanisms underlying resillience to noise-induced tinnitus. One of the main causes of tinnitus is prolonged or repeated exposure to excessive noise. However, not everyone with such exposure develops tinnitus. Certain individuals appear to show a natural resilience. Identifying the basis of this resilience could make it possible to develop drugs that enhance these mechanisms, and thereby extend this protection to those who would otherwise be at risk of tinnitus. Li et al. have brought this a step closer by studying tinnitus resilience mechanisms in mice. Previous work by the same group revealed that in mice with tinnitus a group of neurons in the brainstem called fusiform cells are overly active. These cells receive direct input from the ear, and their hyperactivity is largely due to ion channels called KCNQ2/3 potassium channels being less active. Li et al. now show that exposure to excessive noise causes a reduction in KCNQ2/3 activity in the exposed mice. However, in animals that successfully avoid developing tinnitus, KCNQ2/3 activity spontaneously recovers over the course of a few days. This recovery triggers a reduction in the activity of another type of ion channel, known as the HCN channel. The combined plasticity of KCNQ and HCN channels prevents tinnitus-associated hyperactivity in the fusiform cells. These results suggest that drugs that increase activity of KCNQ2/3 channels, and/or reduce activity of HCN channels, could thus boost resilience to tinnitus. In the future, targeting both channel types at the same time could provide an effective treatment with minimal side effects.

June 2015. In a recent article in the Journal of Neuroscience, Bopanna Kalappa, a postdoctoral fellow in Dr. Tzounopoulos' laboratory reported the discovery of a novel KCNQ2/3-Specific channel activator that suppresses in vivo epileptic activity and prevents the development of tinnitus. KCNQ channels are voltage-dependent potassium channels that are activated at resting membrane potentials and therefore provide a powerful brake on neuronal excitability. Genetic or experience-dependent reduction of KCNQ2/3 channel activity is linked with disorders that are characterized by neuronal hyperexcitability, such as epilepsy and tinnitus. Retigabine, a small molecule that activates KCNQ2–5 channels by shifting their voltage-dependent opening to more negative voltages, is an approved anti-epileptic drug. However, recently identified side effects have limited its clinical use. As a result, the development of improved KCNQ2/3 channel activators is crucial for the treatment of hyperexcitability-related disorders. By incorporating a fluorine substituent in the 3-position of the tri-aminophenyl ring of retigabine, Dr. Tzounopoulos' group, in collaboration with SciFluor and Dr. Tzingounis' group, they synthesized a small-molecule activator (SF0034) with novel properties. Heterologous expression of KCNQ2/3 channels in HEK293T cells showed that SF0034 was five times more potent than retigabine at shifting the voltage dependence of KCNQ2/3 channels to more negative voltages. Moreover, unlike retigabine, SF0034 did not shift the voltage dependence of either KCNQ4 or KCNQ5 homomeric channels. Behavioral studies demonstrated that SF0034 was a more potent and less toxic anticonvulsant than retigabine in rodents. Furthermore, SF0034 prevented the development of tinnitus in mice. SF0034 is not only a powerful tool for investigating ion channel properties, but, most importantly, it provides a clinical candidate for treating epilepsy and preventing tinnitus. Clinical trials to test the efficacy of SF0034 in humans are expected to start soon.

May 2015. In a recent article in the journal of Proceedings of the National Academy of Sciences (PNAS), Charles Anderson, a postdoctoral fellow with Dr. Tzounopoulos, reported that synaptic and tonic zinc modulate extrasynaptic glutamate NMDA-type receptors (NMDARs). As an essential element for living organisms, zinc is a cofactor in many enzymes and regulatory proteins. After the surprising discovery of mobile zinc in synaptic vesicles throughout many areas of the brain, numerous investigators have studied its possible roles during neurotransmission. Nonetheless, knowledge of the physiology of zinc at the synapse is still in its infancy. This study showed that synaptic and tonic zinc inhibit extrasynaptic NMDARs, which are widely distributed in the central nervous system and important for normal and pathological excitatory signaling. This work indicates that the newly discovered interaction between zinc and extrasynaptic NMDARs can provide a general mechanism for controlling neuronal excitability in the central nervous system (CNS).

 

February 2015. In a recent article in the Journal of Neuroscience, Ankur Joshi, a graduate student in Dr. Tzounopoulos' laboratory, reported that cell-specific intrinsic and synaptic mechanisms divide intracortical synaptic excitation from Layer 2/3 (L2/3) to L5B into two functionally distinct pathways with different input–output functions. Because different L5B neurons project either to lower auditory processing centers or to the contalateral cortex, these results determine key cellular synaptic and circuit properties of top-down and cortico-cortical neurons. These results have generated novel hypotheses about the mechanisms underlying responses to sound in auditory cortex and the molecular/cellular rules mediating top-down modulation of auditory processing.

 

Jan 2015. Dr. Srivatsun Sadagopan has joined the Auditory Research Group as its newest faculty member. Srivatsun, who goes by Vatsun, joins us after graduate studies at Johns Hopkins and postdoctoral stints at Northwestern and Rockefeller. Vatsun will study the neuronal mechanisms by which neurons in the auditory cortex maintain stable representations of behaviorally important sounds under varying listening conditions.

 

November 2014. In a recent article from the Kandler-lab in the Journal of Neuroscience, MSTP student Joshua Sturm and former postdoctoral fellow Tuan Nguyen shed new light on how neurons in an important auditory midbrain center, the inferior colliculus, connect to each other. Using laser-scanning photostimulation with caged glutamate they revealed the spatial organization, strength, and developmental changes of excitatory and inhibitory local connections in the mouse inferior colliculus. Results from their studies illustrate important principles pertaining to how the brain processes auditory information and provide insight into biological mechanisms that may underlie disorders of auditory dysfunction such as developmental dyslexia and tinnitus.

July 2014. Dr. Rubio co-authored one manuscript in Frontiers in Neuroscience. entitled: Contribution of short-latency auditory inputs to the neuronal substrates underlying the acoustic startle reflex. In this study, Dr. Rubio and co-authors investigated the anatomical origin and functional role of these inputs using a multidisciplinary approach that combines morphological, electrophysiological and behavioral techniques.This study contributes to understand better the role of neuronal mechanisms in auditory alerting behaviors. Rubio ME, Fukazawa Y, Kamasawa N, Clarkson C, Molnár E, Shigemoto R (2014) Target- and input-dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus J Comp Neurol Jul 19. doi: 10.1002/cne.23654. [Epub ahead of print]. 522:4023-4042. PMID: 25041792

 

July 2014. Dr. Rubio published one manuscript in the Journal of Comparative Neurology. Target- and input-dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus. Using freeze-fracture immunogold labeling for AMPA and NMDA receptors at four distinct types of excitatory synapses in cochlear nucleus, Dr. Rubio and co-authors revealed target- and input-dependent features in the structure, number, and organization of AMPA and NMDA receptors in auditory nerve and parallel fiber synapses. Gómez-Nieto R, de Anchieta J, Horta-Júnior J, Castellano O, Millian-Morell L, Rubio ME, López DE (2014) Contribution of short-latency auditory inputs to the neuronal substrates underlying the acoustic startle reflex. Frontiers in Neuroscience. Published: 25 July 2014 doi: 10.3389/fnins.2014.00216. PMID: 25120419. PMCID: PMC4110630

July 2014.  Dr. Maria Rubio was elected Member of the Scientific Advisory Committee (SAC) of the American Tinnitus Association.

June 2014.  In a recent article in the journal Neuron, the members of the Kandler lab provided the first evidence that the pattern of spontaneous neuronal activity, which is present before hearing onset, plays a crucial role in the precise wiring of central auditory circuits. It has long been known that the immature cochlea spontaneously generates a stereotypical pattern of rhythmic activity even before airborne sound can elicit cochlea responses. But the function of this activity has remained speculative.  To address this question, the authors investigated the structural and functional development of a sound localization pathway in a genetically modified mouse in which developing auditory neurons fire in a different rhythm. By using a combination of sophisticated anatomical and physiological techniques, they were able to demonstrate that these mice have impairments in the strengthening of inhibitory synapses and in the precision of tonotopy, the fundamental organizational principle of auditory circuits. Their results highlight the important role of activity-dependent refinement in the establishment of precise auditory circuits and may help to understand the developmental causes of central auditory processing disorders, which is estimated to affect about 5% of school children. The contributing authors are Amanda Clause and Gunsoo Kim, former graduate students, Cat Weisz, postdoctoral fellow,  Douglas Vetter (University of Mississippi Medical Center), and Mandy Sonntag and Rudolf Rűbsamen (University of Leipzig, Germany). 

May 2014. Dr. Maria Rubio has been selected as a regular member of the Auditory Study Section (AUD) of the National Institute of Health (NIH).

May 2014. Joshua Sturm, MSTP graduate student in Dr. Kandler's group, was awarded an Individual National Research Service Dual-Doctoral Degree Fellowship from the National Institute on Deafness and other Communication Disorders. Under this 4-year award, Joshua Sturm will use laser scanning photostimulation to investigate how neuronal connections in the auditory midbrain change in a mouse model of tinnitus

January 2014. Postdoctoral fellow Dr. Charles Anderson received a National Research Service Award (NRSA) from the National Institute on Deafness and Other Communication Disorders. Dr. Anderson is a member of the laboratory of Thanos Tzounopoulos and this award will support his investigations towards understanding the role of synaptic zinc in normal and pathological (tinnitus) auditory processing.

June 2013. Dr. Jason Middleton, a postdoctoral fellow with Dr. Tzounopoulos, has just started his independent career as a tenure-track Assistant Professor at the Department of Cell Biology and Anatomy, LSU Health Sciences Center - New Orleans. Dr. Middleton will continue working on auditory circuits in his own laboratory.

June 2013. In a recent article in the journal of Proceedings of the National Academy of Sciences (PNAS), Shuang Li, a graduate student with Dr. Tzounopoulos, reported that a reduction in Kv7 (KCNQ) potassium channel activity is essential for the induction of tinnitus. This reduction is due to a shift in the voltage dependence of Kv7 channel activation to more positive voltages. In vivo studies demonstrate that a pharmacological manipulation that shifts the voltage dependence of Kv7 to more negative voltages prevents the development of tinnitus. This study provides an important link between the biophysical properties of the Kv7 channel and the generation of tinnitus. Importantly, this study points to novel biological targets for designing therapeutic drugs that may prevent the development of tinnitus in humans (Pathogenic Plasticity of Kv7.2/3 Channel Activity is Essential for the Induction of Tinnitus. Shuang Li, Veronica Choi and Thanos Tzounopoulos.PNAS, 2013, June 11; 110(24): 9980-9985).

May 2013. In a recent article in the Journal of Neuroscience, Dr. Perez-Rosello, a postdoctoral fellow with Dr. Tzounopoulos, reported a new and unexpected action of zinc in the dorsal cochlear nucleus (DCN). Although it is well established that many glutamatergic neurons sequester Zn2+ within their synaptic vesicles, the physiological significance of synaptic Zn2+ remains poorly understood.  In mouse brainstem slices, release of synaptic Zn2+ reduced evoked release probability at parallel fiber terminals, reducing EPSC amplitudes in fusiform neurons of the DCN. Surprisingly, zinc also increased synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in DCN, and the effects of zinc on synaptic transmission were blocked by inhibiting 2-AG synthesis or antagonizing cannabinoid CB1 receptors on parallel fiber terminals. Altogether, the results suggest that Zn2+ activates postsynaptic mZnRs, triggering synthesis of endocannabinoids that reduce release probability by acting on presynaptic CB1 receptors. This article was selected by the Editors of J. of Neuroscience as a featured article that was highlighted at “This Week in the Journal”.

February 2013.  Dr. Catherine Weisz, a postdoctoral fellow with Dr. Kandler, received a National Research Service Award (NRSA) from the National Institute on Deafness and Other Communication Disorders. This award ($101,400 for two years) will support Dr. Weisz' research in investigating the synaptic mechanisms involved in the plasticity of developing auditory connections.

August 2012. Dr. Jason Castro, postdoctoral fellow with Dr. Kandler, accepted a faculty position as Assistant professor in the Department of Neuroscience at Bates College. Congratulations and best luck in you future career!

July, 2012.  Dr. Tuan Nguyen, postdoctoral fellow with Dr. Kandler, accepted a faculty position as Assistant professor in the Department of Physics at The College of New Jersey.  Congratulations and best luck in you future career!

June 2012.  Dr. Jason Middelton -- postdoctoral fellow with Dr.Tzounopoulos -- received an R03 grant award from NIDCD. This award will support his investigations towards understanding the microcircuit organization of cortical top-down auditory pathways.

August 2011.  Postdoctoral fellow Dr. Jason Castro received a National Research Service Award (NRSA) from the National Institute on Deafness and Other Communication Disorders. This fund will support Dr. Castro, a member of Karl Kandler’s laboratory, to investigate novel cellular mechanisms that regulate the strength of neuronal connection in the auditory system.

July 2011. Postdoctoral fellow Dr. Tamara Perez-Rosello received a National Research Service Award (NRSA) from the National Institute on Deafness and Other Communication Disorders. Dr. Perez-Rosello is a member of the laboratory of Thanos Tzounopoulos and this award will support her investigations towards understanding the role of synaptic zinc in the auditory brainstem.

July 2011.  Karl Kandler and Bill Yates were awarded an Institutional Training Grant (T32) from the National Institute on Deafness and Other Communication Disorders. This five-year award provides funds to support training of students and postdoctoral fellows in the auditory and vestibular neuroscience.

July 2011. Dr. Karl Kandler became the Chair of the Auditory Systems (AUD) National Institute of Health (NIH) study section.

In a recent article in the journal of Proceedings of the National Academy of Sciences (PNAS), Dr. Tzounopoulos and associates provide novel evidence about the cellular mechanisms that underlie hyperactivity in auditory brainstem circuits in mice with behavioral evidence of tinnitus. This study makes use of novel imaging and behavioral techniques to demontrate that a decrease in GABAergic inhibition undrelies pathological hyperxcitability that is observed in the auditory braisntem of mice with behavioral evidence of tinnitus. Besides its implications towards understanding the biology of tinnitus, this study may eventually pave the way towards developing novel drug-based treatments of tinnitus in humans.

In a recent article in the Journal of Neuroscience, Zhao and Tzounopoulos determined that physiological activation of cholinergic inputs controls associative synaptic plasticity in the dorsal cochler nucleus. This modulation occurs via activation of endocannabinoid signaling. This study reveals the importance of cholinergic modulation on the plasticity mechanisms found in the auditory brainstem.

In a recent article in the journal Neuroscience, Ricardo Gómez-Nieto and María Rubio provide a detailed description of the fine structure and adjacent neuropil of the main projection neurons (bushy cells) in the anteroventral cochlear nucleus of the rhesus monkey. These data provide evidence of a similar synaptic organization for the bushy cells in primate and other mammals  (Gómez-Nieto R, Rubio M.E, Ultrastructure, synaptic organization, and molecular components of bushy cell networks in the anteroventral cochlear nucleus of the rhesus monkey. Neuroscience, In Press).

In a recent article in the journal Neuroscience, Kim and Kandler characterized the synaptic mechanism by which inhibitory connections in an inhibitory sound localization pathway become strengthened during development  (Kim G, Kandler K, Synaptic changes underlying the strengthening of GABA/glycinergic connections in the developing lateral superior olive. Neuroscience 171:924-33)